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Aim: To investigate the accuracy of the strain histogram endoscopic ultrasound (EUS)-based method for the diagnostic differentiation of patients with pancreatic masses., Methods: In a prospective single center study, 149 patients were analyzed, 105 with pancreatic masses and 44 controls. Elastography images were recorded using commercially available ultrasound equipment in combination with EUS linear probes. Strain histograms (SHs) were calculated by machine integrated software in regions of interest and mean values of the strain histograms were expressed as Mode 1 (over the mass) and Mode 2 (over an adjacent part of pancreatic tissue, representing the reference area). The ratio between Mode 2 and Mode 1 was calculated later, representing a new variable, the strain histogram ratio. After the final diagnosis was established, two groups of patients were formed: a pancreatic cancer group with positive cytology achieved by fine needle aspiration puncture or histology after surgery (58 patients), and a mass-forming pancreatitis group with negative cytology and follow-up after 3 and 6 mo (47 patients). All statistical analyses were conducted in SPSS 14.0 (SPSS Inc., Chicago, IL, United States)., Results: Results were obtained with software for strain histograms with reversed hue scale (0 represents the hardest tissue structure and 255 the softest). Based on the receiver operating characteristics (ROC) curve coordinates, the cut-off point for Mode 1 was set at the value of 86. Values under the cut-off point indicated the presence of pancreatic malignancy. Mode 1 reached 100% sensitivity and 45% specificity with overall accuracy of 66% (95%CI: 61%-66%) in detection of pancreatic malignant tumors among the patients with pancreatic masses. The positive and negative predictive values were 54% and 100%, respectively. The cut-off for the new calculated variable, the SH ratio, was set at the value 1.153 based on the ROC curve coordinates. Values equal or above the cut-off value were indicative of pancreatic malignancy. The SH ratio reached 98% sensitivity, 50% specificity and an overall accuracy of 69% (95%CI: 63%-70%). The positive and negative predictive values were 92% and 100%, respectively., Conclusion: SH showed high sensitivity in pancreatic malignant tumor detection but disappointingly low specificity. Slight improvements in specificity and accuracy were achieved using the SH ratio.